RESUMEN
BACKGROUND: Epidermolysis bullosa simplex generalized severe (EBS-gen sev) is a genetic disorder caused by mutation in the KRT5 or KRT14 genes. Although it is usually considered a mechanical disease, recent data argue for additional inflammatory mechanisms. OBJECTIVES: To assess the inflammation in the skin of patients with EBS-gen sev. METHODS: A first immunohistochemical retrospective study was performed on frozen skin samples from 17 patients with EBS-gen sev. A second multicentre prospective study was conducted on 10 patients with severe EBS-gen sev. Blister fluid and epidermis were processed for immunochemical analysis and quantitative real-time polymerase chain reaction. Cytokine expression was analysed in blister fluid and compared with that in controls. RESULTS: Histological analysis showed a constant dermal perivascular CD4+ lymphocyte infiltrate in skin biopsies of both blister (n = 17) and rubbed skin (n = 5), an epidermal infiltration of neutrophils and eosinophils in 70% of cases, and increased immunostaining for CXCL9 and CXCL10 in blistering skin. High levels of T helper 17 cytokines were detected in lesional skin. Three adult patients with EBS-gen sev were treated with apremilast, with a dramatic improvement of skin blistering and good tolerance. CONCLUSIONS: Our study demonstrates the importance of inflammation in patients with EBS-gen sev and underlines the key role for T helper 17 cells in its pathogenesis. In addition, this study provides promising new therapeutic approaches for this disabling disorder.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Epidermólisis Ampollosa Simple/inmunología , Piel/efectos de los fármacos , Células Th17/inmunología , Talidomida/análogos & derivados , Adulto , Antiinflamatorios no Esteroideos/uso terapéutico , Niño , Preescolar , Epidermólisis Ampollosa Simple/tratamiento farmacológico , Epidermólisis Ampollosa Simple/genética , Femenino , Humanos , Lactante , Recién Nacido , Queratina-14/genética , Queratina-5/genética , Masculino , Persona de Mediana Edad , Mutación , Proyectos Piloto , Estudios Retrospectivos , Piel/citología , Piel/inmunología , Células Th17/efectos de los fármacos , Talidomida/farmacología , Talidomida/uso terapéutico , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Currently, there are no histological criteria to diagnose irritable bowel syndrome (IBS). Our aims were (i) to examine the distribution of inflammatory cells in the colon of healthy and IBS subjects and (ii) to find histological diagnosis criteria for IBS. METHODS: Colonic biopsies were taken from four distinct regions of the colon from 20 controls (HC) and 11 patients with IBS (4 with constipation (IBS-C) and 7 with diarrhea (IBS-D) and embedded in paraffin. Macrophages, mast cells, eosinophils, and T lymphocytes were immunostained and positive cells counted. KEY RESULTS: In both HC and IBS patients, global cellularity decreased from the cecum to the rectum (P < .01) which is attributed to reduced number of macrophages (P < .05) and eosinophils (P < .001) but not T cells. Mast cells were reduced in IBS (P < .05) but not in HC, particularly in IBS-D (P < .05). Results showed higher number of macrophages in the left colon of IBS subjects than HC (P < .05). CONCLUSION & INFERENCES: Here we report a decreasing gradient of immune cells from the cecum to the rectum of the human colon. Although global cellularity cannot be used to distinguish between IBS and HC, closer analysis of macrophages and mast cells may be useful markers to confirm IBS histologically and to differentiate between IBS-C and IBS-D when clinical presentation alternates between constipation and diarrhoea. This pilot study remains to be confirmed with greater number of patients.
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Colon/inmunología , Inflamación/inmunología , Mucosa Intestinal/inmunología , Síndrome del Colon Irritable/diagnóstico , Síndrome del Colon Irritable/inmunología , Anciano , Biopsia , Colon/patología , Eosinófilos/patología , Femenino , Humanos , Inflamación/complicaciones , Inflamación/patología , Mucosa Intestinal/patología , Síndrome del Colon Irritable/complicaciones , Síndrome del Colon Irritable/patología , Macrófagos/patología , Masculino , Mastocitos/patología , Persona de Mediana Edad , Proyectos Piloto , Linfocitos T/patologíaRESUMEN
The mucosal immune system (including airway, intestinal, oral and cervical epithelium) is an integrated network of tissues, cells and effector molecules that protect the host from environmental insults and infections at mucous membrane surfaces. Dysregulation of immunity at mucosal surfaces is thought to be responsible for the alarming global increase in mucosal inflammatory diseases such as those affecting the gastrointestinal (Crohn's disease, ulcerative colitis and irritable bowel syndrome) and respiratory (asthma, allergy and chronic obstructive pulmonary disorder) system. Although immune regulation has been well-studied in isolated mucosal sites, the extent of the immune interaction between anatomically distant mucosal sites has been mostly circumstantial and the focus of much debate. With novel technology and more precise tools to examine histological and functional changes in tissues, today there is increased appreciation of the 'common mucosal immunological system' originally proposed by Bienenstock nearly 40 years ago. Evidence is amounting which shows that stimulation of one mucosal compartment can directly and significantly impact distant mucosal site, however the mechanisms are unknown. Today, we are only beginning to understand the complexity of relationships and communications that exist between different mucosal compartments. A holistic approach to studying the mucosal immune system as an integrated global organ is imperative for future advances in understanding mucosal immunology and for future treatment of chronic diseases. In this review, we particularly focus on the latest evidence and the mechanisms operational in driving the lung-gut cross-talk.
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Retroalimentación Fisiológica , Intestinos/fisiología , Pulmón/fisiología , Animales , Humanos , Hipersensibilidad/etiología , Hipersensibilidad/metabolismo , Hipersensibilidad/patología , Inmunidad Mucosa , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Membrana Mucosa/patología , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patologíaRESUMEN
Increased risk of allergy during early life indicates deficient immune regulation in this period of life. To date, the cause for inefficient neonatal immune regulation has never been elucidated. We aimed to define the ontogeny of oral tolerance and to identify necessary conditions specific for this stage of life. Ovalbumin (OVA) was administered orally to mice through breast milk and efficiency of systemic tolerance to OVA was assessed in adulthood using a model of allergic airway inflammation. Oral tolerance induction was fully efficient starting third week of life. Inefficiency in neonates was a consequence of abnormal antigen transfer across the gut barrier and retinaldehyde dehydrogenase expression by mesenteric lymph node CD103(+) neonatal dendritic cells, resulting in inefficient T-cell activation. Neonates' serum retinol levels were three times lower than in adult mice, and vitamin A supplementation was sufficient to rescue neonatal defects and allow tolerance induction from birth. The establishment of oral tolerance required the differentiation of Th1 lymphocytes in both vitamin A-supplemented neonates and 3-week-old unsupplemented mice. This knowledge should guide the design of interventions for allergy prevention that are adapted to the neonatal stage of life such as vitamin A supplementation.
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Tolerancia Inmunológica/efectos de los fármacos , Ovalbúmina/farmacología , Células TH1/inmunología , Deficiencia de Vitamina A/prevención & control , Vitamina A/administración & dosificación , Administración Oral , Animales , Animales Recién Nacidos , Animales Lactantes , Antígenos CD/genética , Antígenos CD/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Expresión Génica , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Mesenterio/citología , Mesenterio/inmunología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Células TH1/citología , Vitamina A/inmunología , Vitamina A/metabolismo , Deficiencia de Vitamina A/inmunología , Deficiencia de Vitamina A/fisiopatologíaRESUMEN
BACKGROUND: Gut microbiome patterns have been associated with predisposition to eczema potentially through modulation of innate immune signalling. OBJECTIVE: We examined gut microbiome development in the first year of life in relation to innate immune responses and onset of IgE-associated eczema over the first 2.5 years in predisposed children due to maternal atopy [www.anzctr.org.au, trial ID ACTRN12606000280505]. METHODS: Microbial composition and diversity were analysed with barcoded 16S rRNA 454 pyrosequencing in stool samples in pregnancy and at ages 1 week, 1 month and 12 months in infants (n = 10) who developed IgE-associated eczema and infants who remained free of any allergic symptoms at 2.5 years of age (n = 10). Microbiome data at 1 week and 1 month were analysed in relation to previously assessed immune responses to TLR 2 and 4 ligands at 6 months of age. RESULTS: The relative abundance of Gram-positive Ruminococcaceae was lower at 1 week of age in infants developing IgE-associated eczema, compared with controls (P = 0.0047). At that age, the relative abundance of Ruminococcus was inversely associated with TLR2 induced IL-6 (-0.567, P = 0.042) and TNF-α (-0.597, P = 0.032); there was also an inverse association between the abundance of Proteobacteria (comprising Gram-negative taxa) and TLR4-induced TNF-α (rs = -0.629, P = 0.024). This relationship persisted at 1 month, with inverse associations between the relative abundance of Enterobacteriaceae (within the Proteobacteria phylum) and TLR4-induced TNF-α (rs = -0.697, P = 0.038) and Enterobacteriaceae and IL-6 (rs = -0.709, P = 0.035). Mothers whose infants developed IgE-associated eczema had lower α-diversity of Bacteroidetes (P = 0.04) although this was not seen later in their infants. At 1 year, α-diversity of Actinobacteria was lower in infants with IgE-associated eczema compared with controls (P = 0.002). CONCLUSION AND CLINICAL RELEVANCE: Our findings suggest that reduced relative abundance of potentially immunomodulatory gut bacteria is associated with exaggerated inflammatory cytokine responses to TLR-ligands and subsequent development of IgE-associated eczema.
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Dermatitis Atópica/inmunología , Bacterias Grampositivas/inmunología , Inmunidad Innata , Inmunoglobulina E/inmunología , Intestinos/microbiología , Exposición Materna/efectos adversos , Preescolar , Dermatitis Atópica/microbiología , Susceptibilidad a Enfermedades , Femenino , Bacterias Grampositivas/clasificación , Bacterias Grampositivas/aislamiento & purificación , Humanos , Lactante , Interleucina-6/inmunología , Intestinos/inmunología , Masculino , Embarazo , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVE: To determine the role of colonic barrier defects and low-grade inflammation in irritable bowel syndrome (IBS)-like symptoms in quiescent inflammatory bowel disease (IBD). DESIGN: Caecal biopsies were collected from 51 IBS, 49 quiescent IBD (31 Crohn's disease (CD) and 18 ulcerative colitis (UC)) patients and 27 controls. IBS was assessed using the Rome III criteria and the IBS severity score. Epithelial barrier integrity was evaluated by determining the paracellular permeability of biopsies mounted in Ussing chambers and the mRNA expression of tight junction proteins (ZO-1, α-catenin and occludin). Low-grade inflammation was evaluated by counting cells, including intraepithelial lymphocytes (IELs), eosinophils and mast cells, and by determining the mRNA and protein expression of tumour necrosis factor (TNF)-α in biopsies and culture supernatants. RESULTS: IBS-like symptoms were present in 35.4 and 38% of CD and UC patients, respectively. Paracellular permeability was significantly increased in both quiescent IBD with IBS-like symptoms and IBS compared with quiescent IBD without IBS-like symptoms (p<0.01, respectively) or controls (p<0.01, respectively). Significantly lower expression of ZO-1 and α-catenin was detected in IBS and quiescent IBD with IBS-like symptoms. IELs and TNF-α were significantly increased in quiescent IBD with IBS-like symptoms, but not in IBS. CONCLUSIONS: In quiescent IBD, IBS-like symptoms related to persistent subclinical inflammation associated with increased colonic paracellular permeability. A persistent increase in TNF-α in colonic mucosa may contribute to the epithelial barrier defects associated with abdominal pain in quiescent IBD, but not in IBS. Optimisation of anti-inflammatory therapy may be considered in quiescent IBD with IBS-like symptoms.
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Colitis Ulcerosa/complicaciones , Colon/metabolismo , Enfermedad de Crohn/complicaciones , Mucosa Intestinal/metabolismo , Síndrome del Colon Irritable/etiología , Adulto , Anciano , Biomarcadores/metabolismo , Estudios de Casos y Controles , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/metabolismo , Colon/inmunología , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Femenino , Humanos , Inmunohistoquímica , Mucosa Intestinal/inmunología , Síndrome del Colon Irritable/inmunología , Síndrome del Colon Irritable/metabolismo , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Permeabilidad , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Índice de Severidad de la Enfermedad , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
There is an urgent need to identify environmental risk and protective factors in early life for the prevention of allergy. Our study demonstrates the presence of respiratory allergen from house dust mite, Der p 1, in human breast milk. Der p 1 in milk is immunoreactive, present in similar amounts as dietary egg antigen, and can be found in breast milk from diverse regions of the world. In a mouse model of asthma, oral exposure to Der p through breast milk strongly promotes sensitization rather than protect the progeny as we reported with egg antigen. These data highlight that antigen administration to the neonate through the oral route may contribute to child allergic sensitization and have important implications for the design of studies assessing early oral antigen exposure for allergic disease prevention. The up-to-now unknown worldwide presence of respiratory allergen in maternal milk allows new interpretation and design of environmental control epidemiological studies for allergic disease prevention.
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Alérgenos/inmunología , Asma/inmunología , Leche Humana/inmunología , Pyroglyphidae/inmunología , Animales , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/inmunología , Calostro/inmunología , Cisteína Endopeptidasas/inmunología , Exposición a Riesgos Ambientales/efectos adversos , Femenino , Humanos , EmbarazoRESUMEN
BACKGROUND AND OBJECTIVE: Relative deficiency of dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) has been implicated in the rising allergy prevalence in Westernized countries. Fish oil supplementation may provide an intervention strategy for primary allergy prevention. The objective of this study was to assess the effect of fish oil n-3 PUFA supplementation from birth to 6 months of age on infant allergic disease. METHODS: In a double-blind randomized controlled trial, 420 infants at high atopic risk received a daily supplement of fish oil containing 280 mg docosahexaenoic acid and 110 mg eicosapentaenoic acid or a control (olive oil), from birth to age 6 months. PUFA levels were measured in 6-month-old infants' erythrocytes and plasma and their mothers' breast milk. Eczema, food allergy, asthma and sensitization were assessed in 323 infants for whom clinical follow-up was completed at 12 months of age. RESULTS: At 6 months of age, infant docosahexaenoic acid and eicosapentaenoic acid levels were significantly higher (both P < .05) and erythrocyte arachidonic acid levels were lower (P = .003) in the fish oil group. Although n-3 PUFA levels at 6 months were associated with lower risk of eczema (P = .033) and recurrent wheeze (P = .027), the association with eczema was not significant after multiple comparisons and there was no effect of the intervention per se on the primary study outcomes. Specifically, between-group comparisons revealed no differences in the occurrence of allergic outcomes including sensitization, eczema, asthma, or food allergy. CONCLUSIONS: Postnatal fish oil supplementation improved infant n-3 status but did not prevent childhood allergic disease.
Asunto(s)
Suplementos Dietéticos , Aceites de Pescado/uso terapéutico , Hipersensibilidad Inmediata/prevención & control , Biomarcadores/metabolismo , Ácidos Docosahexaenoicos/sangre , Ácidos Docosahexaenoicos/uso terapéutico , Esquema de Medicación , Ácido Eicosapentaenoico/sangre , Ácido Eicosapentaenoico/uso terapéutico , Femenino , Estudios de Seguimiento , Humanos , Hipersensibilidad Inmediata/sangre , Hipersensibilidad Inmediata/diagnóstico , Lactante , Recién Nacido , Análisis de Intención de Tratar , Modelos Logísticos , Masculino , Leche Humana/metabolismo , Riesgo , Pruebas Cutáneas , Resultado del TratamientoRESUMEN
BACKGROUND: Maternal fish oil supplementation during pregnancy has been associated with altered infant immune responses and a reduced risk of infant sensitization and eczema. OBJECTIVE: To examine the effect of early postnatal fish oil supplementation on infant cellular immune function at 6 months of age in the context of allergic disease. METHODS: In a double-blind randomized controlled trial (ACTRN12606000281594), 420 infants of high atopic risk received fish oil [containing 280 mg docosahexaenoic acid (DHA) and 110 mg eicosapentanoic acid (EPA)] or control oil daily from birth to 6 months. One hundred and twenty infants had blood collected at 6 months of age. Fatty acid levels, induced cytokine responses, T cell subsets and monocyte HLA-DR expression were assessed at 6 months of age. Infant allergies were assessed at 6 and 12 months of age. RESULTS: DHA and EPA levels were significantly higher in the fish oil group and erythrocyte arachidonic acid (AA) levels were lower (all P < 0.05). Infants in the fish oil group had significantly lower IL-13 responses (P = 0.036) to house dust mite (HDM) and higher IFNγ (P = 0.035) and TNF (P = 0.017) responses to phytohaemaglutinin (PHA). Infants with relatively high DHA levels had lower Th2 responses to allergens including lower IL-13 to ß-lactoglobulin (BLG) (P = 0.020), and lower IL-5 to BLG (P = 0.045). CONCLUSIONS AND CLINICAL RELEVANCE: Postnatal fish oil supplementation increased infant n-3 polyunsaturated fatty acid (PUFA) levels and associated with lowered allergen-specific Th2 responses and elevated polyclonal Th1 responses. Our results add to existing evidence of n-3 PUFA having immunomodulatory properties that are potentially allergy-protective.
Asunto(s)
Suplementos Dietéticos , Aceites de Pescado/farmacología , Inmunidad/efectos de los fármacos , Factores Inmunológicos/farmacología , Inmunidad Adaptativa/efectos de los fármacos , Factores de Edad , Citocinas/biosíntesis , Citocinas/inmunología , Ácidos Grasos Insaturados/sangre , Femenino , Aceites de Pescado/administración & dosificación , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/inmunología , Inmunidad Innata/efectos de los fármacos , Factores Inmunológicos/administración & dosificación , Lactante , MasculinoRESUMEN
BACKGROUND: Dietary changes may epigenetically modify fetal gene expression during critical periods of development to potentially influence disease susceptibility. This study examined whether maternal and/or fetal folate status in pregnancy is associated with infant allergic outcomes. METHODS: Pregnant women (n=628) were recruited in the last trimester of pregnancy. Folate status determined by both food frequency questionnaires and folate levels in maternal and cord blood serum was examined in relation to infant allergic outcomes at 1 year of age (n=484). RESULTS: Infants who developed allergic disease (namely eczema) did not show any differences in cord blood or maternal folate levels compared with children without disease. Although maternal folate intake from foods was also not different, folate derived from supplements was higher (P=0.017) in children with subsequent eczema. Furthermore, infants exposed to >500 µg folic acid/day as a supplement in utero were more likely to develop eczema than those taking <200 µg/day (OR [odds ratio] =1.85; 95% CI 1.14-3.02; P=0.013), remaining significant after adjustment for maternal allergy and other confounders. There was a nonlinear relationship between cord blood folate and sensitization, with folate levels <50 nmol/l (OR=3.02; 95% CI 1.16-7.87; P=0.024) and >75 nmol/l (OR=3.59; 95% CI 1.40-9.20; P=0.008) associated with greater sensitization risk than levels between 50 and 75 nmol/l. CONCLUSION: Fetal levels between 50 and 75 nmol/l appeared optimal for minimizing sensitization. While folate taken as a supplement in higher doses during the third trimester was associated with eczema, there was no effect on other allergic outcomes including sensitization. Further studies are needed to determine the significance of this.
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Sangre Fetal/química , Ácido Fólico/sangre , Hipersensibilidad/etiología , Embarazo/sangre , Efectos Tardíos de la Exposición Prenatal/sangre , Adulto , Dieta , Suplementos Dietéticos/efectos adversos , Femenino , Humanos , Lactante , Recién NacidoRESUMEN
BACKGROUND: Structural cells are an important reservoir of chemokines that coordinate the influx of various immune cells to the lungs of asthmatics. Airway smooth muscle cells (ASMC) are an important source of these chemokines. CCL15 is a recently described chemo-attractant for neutrophils, eosinophils, monocytes and lymphocytes. OBJECTIVE: To determine the production and the regulation of CCL15 by ASMC and to investigate its production in asthmatic airways. METHODS: Human ASMC were obtained from main bronchial airway segments of patients with mild, moderate and severe asthma. To induce chemokine production, cells were incubated with IL-4, IL-13, TNF-α or IFN-γ in presence or absence of dexamethasone, mithramycin A (SP-1 inhibitor) or the IKK-2 inhibitor, AS602868. CCL15 mRNA expression was evaluated by real-time PCR. Immunoreactive CCL15 was detected by immuno-fluorescence and CCL15 protein concentration in the supernatant was measured using ELISA. RESULTS: CCL15 is constitutively expressed in human ASMC and is strongly up-regulated by TNF-α. This up-regulation is inhibited by dexamethasone, mithramycin A and AS602868. TNF-α-induced CCL15 levels can be synergistically enhanced by the presence of IFN-γ, at both the transcriptional and translation level. This synergism is NF-κB-dependent. Asthmatic biopsies demonstrated higher expression of CCL15 compared with non-asthmatic controls. CONCLUSION AND CLINICAL RELEVANCE: Our results show that ASMC are a potent source of CCL15 in the airways and may directly participate in the recruitment of inflammatory cells to asthmatic airways. Targeting the production of CCL15 by ASMC might reduce the inflammatory response within the airways of asthmatic patients.
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Asma/fisiopatología , Bronquios/citología , Quimiocinas CC/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Miocitos del Músculo Liso/metabolismo , Regulación hacia Arriba , Adulto , Asma/inmunología , Biopsia , Quimiocinas CC/efectos de los fármacos , Quimiocinas CC/genética , Quimiocinas CC/inmunología , Femenino , Humanos , Interferón gamma/inmunología , Interferón gamma/farmacología , Proteínas Inflamatorias de Macrófagos/efectos de los fármacos , Proteínas Inflamatorias de Macrófagos/genética , Proteínas Inflamatorias de Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Índice de Severidad de la Enfermedad , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaAsunto(s)
Sistema Respiratorio/patología , Rinitis Alérgica Perenne/patología , Rinitis Alérgica Estacional/patología , Corticoesteroides/uso terapéutico , Humanos , Sistema Respiratorio/metabolismo , Rinitis Alérgica Perenne/tratamiento farmacológico , Rinitis Alérgica Perenne/metabolismo , Rinitis Alérgica Estacional/tratamiento farmacológico , Rinitis Alérgica Estacional/metabolismoRESUMEN
Nitric oxide synthase 1 (NOS1) is a major determinant of bronchial responsiveness in mice and has been proposed as an asthma gene in man. Nevertheless, how nitric oxide production by NOS1 contributes to airway responsiveness remains unclear. Although NOS1 is usually closely associated with nerves, it has also been found in a variety of other cell types, particularly epithelium. We sought to better understand the role of NOS1 by determining its major site of expression in murine airways. Using nicotinamide adenine dinucleotide phosphate-diaphorase (diaphorase), which non-selectively detects nitric oxide synthase (NOS), we found strong evidence of NOS in the airways largely restricted to the airway epithelium and trachea glands. In contrast, diaphorase staining of NOS1-deficient mutant mice demonstrated a marked reduction in epithelial cells of the trachea but not bronchioles, suggesting that the epithelium is the major site of NOS1 expression. This was supported by immunohistochemistry, which also demonstrated significant staining in glands and to a lesser degree in airway smooth muscle. Double immunofluorescence staining of tracheas for NOS1 and the nerve marker PGP 9.5 failed to demonstrate co-localization, indicating that nerves are not an important source of NOS1 in the murine airway wall. Finally, removal of the trachea epithelium by digestion resulted in a marked decrease in NOS1 detection by Western blotting, confirming the epithelium as the major site of NOS1 expression in the murine airway. These findings support the notion that the role of NOS1 in murine bronchial responsiveness involves the epithelium of the central airways.
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Óxido Nítrico Sintasa de Tipo I/fisiología , Óxido Nítrico Sintasa/metabolismo , Tráquea/metabolismo , Animales , Epitelio/metabolismo , Regulación Enzimológica de la Expresión Génica , Inmunohistoquímica , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , NADPH Deshidrogenasa/metabolismo , ARN Mensajero/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Airway remodelling refers to changes in the airway structure and includes subepithelial fibrosis, increased smooth muscle mass, submucosal gland enlargement, neovascularisation and epithelial alterations. Remodelling is observed in response to chronic injury and is seen not only in asthma but in all airway diseases. Remodelling is associated with more severe airflow obstruction and airway hyperresponsiveness in asthma; however, the clinical significance of this is still a matter of debate. Research should be pursued to better understand the accurate implication of airway remodelling in disease and its therapeutic modulation. To allow research in this field, accurate and standardised methods should be utilised to measure airway alterations in disease and following therapy. The standard detection of structural alterations is through direct analyses of airway tissues obtained during a post mortem, surgically or by flexible bronchoscopy. To avoid invasive techniques, other tools have been developed to indirectly measure remodelling, including induced sputum, bronchoalveolar lavage fluid, blood and urine analyses, physiological and radiological assessments, as well as in vitro techniques. Although of great interest, the exact significance of airway remodelling measurements gained through such indirect techniques is uncertain and further research is needed. Despite their invasive nature, direct methods should be favoured to adequately measure airway remodelling in disease and its modulation by therapy.
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Asma/patología , Enfermedad Pulmonar Obstructiva Crónica/patología , Actinas/metabolismo , Resistencia de las Vías Respiratorias/fisiología , Biopsia , Broncoscopía , Colágeno Tipo III/metabolismo , Fibrosis/patología , Humanos , Metaloproteinasas de la Matriz/metabolismo , Músculo Liso/patología , Neovascularización Patológica/patología , Investigación , Mucosa Respiratoria/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Toll-like receptor 4 (TLR4), part of the bacterial lipopolysaccharide (LPS) receptor, is an important bridge between innate and adaptive immunity. Our previous studies have indicated reduced expression of TLR4 and reduced responsiveness to LPS in nasal mucosa of atopic adults compared with non-atopic adults. IL-4 and signal transducer and activator of transcription 6 (STAT6), which are increased in atopic patients, may have a role in modulating TLR4. OBJECTIVE: To examine direct effects of IL-4 and STAT6 on TLR4 expression of U-937 monocytic cells. METHODS: LPS responsiveness, under different conditions of U-937 cells was measured by nuclear factor (NF)-kappaB activation of transcription. TLR4 mRNA was quantified by real-time PCR and TLR4 surface expression was measured by flow cytometry. The promoter and 4.3 kb of the upstream region of TLR4 were cloned into a plasmid vector and transiently transfected into U-937 cells. Transfected cells were incubated with IL-4 and transcriptional activity was assayed by the luciferase assay. STAT6 was transfected to evaluate overexpression of this transcription factor. Cells were also incubated with Tyrphostin AG490 to inhibit tyrosine kinases. RESULTS: NF-kappaB activation by LPS was inhibited by IL-4 pre-incubation but not when IL-4 was added at the same time as LPS. TLR4 mRNA expression was inhibited by IL-4 as early as 6 h but the effect was lost by 24 h. Surface expression of TLR4 was inhibited by IL-4 at 12 and 24 h, but returned to baseline at 48 h. IL-4 inhibited activity of the TLR4 promoter as early as 6 h, but, like the mRNA, these effects were transient. STAT6 overexpression enhanced the inhibition of the TLR4 promoter and prolonged it. Inhibition of TLR4 by IL-4 was abolished by pre-incubation with the tyrosine kinase inhibitor Tyrphostin AG490. CONCLUSION: Our findings demonstrate that IL-4, through STAT6, can modulate TLR4 expression and suggests that Th2 cytokines can impact on the LPS responsiveness of cells.
Asunto(s)
Regulación hacia Abajo , Monocitos/metabolismo , Factor de Transcripción STAT6/metabolismo , Receptor Toll-Like 4/metabolismo , Adulto , Línea Celular , Citometría de Flujo , Humanos , Interleucina-4/farmacología , Lipopolisacáridos , FN-kappa B/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/análisis , Receptor Toll-Like 4/genética , Transfección/métodos , Tirfostinos/farmacologíaRESUMEN
Asthma was originally described as an inflammatory disease that predominantly involves the central airways. Pathological and physiological evidence reported during the past few years suggests that the inflammatory process extends beyond the central airways to the peripheral airways and the lung parenchyma. The small airways are capable of producing T-helper-2 cytokines, as well as chemokines, and they have recently been recognized as a predominant site of airflow obstruction in asthmatic persons. The inflammation at this distal site has been described as more severe than large airway inflammation. These findings are of great clinical significance, and highlight the need to consider the peripheral airways as a target in any therapeutic strategy for treatment of asthma.
Asunto(s)
Asma/complicaciones , Bronquitis/complicaciones , Animales , Asma/patología , Asma/fisiopatología , Asma/terapia , Bronquitis/patología , Bronquitis/fisiopatología , Humanos , Pulmón/patología , Pulmón/fisiopatologíaRESUMEN
We have previously shown that exposure of sensitized animals to lipopolysaccharide (LPS) 18 h after ovalbumin (OVA) challenge inhibits late-airway response (LAR). Using relatively selective nitric oxide synthase (NOS) inhibitors we have shown that LPS upregulates inducible NOS (iNOS) and downregulates constitutive NOS (cNOS) activity. In this study we set out to quantitate NOS isoenzyme activity in lung homogenates and to measure ex vivo interleukin (IL)-10 in tracheal explants of naive or sensitized and OVA-challenged rats exposed to LPS. iNOS activity was increased and cNOS activity reduced 6 h after LPS exposure in naive animals (n = 6, P < 0.001) and at 18 (n = 5, P < 0.001) or 24 (n = 5, P < 0.001) h after OVA challenge in sensitized animals. LPS exposure 18 h after OVA challenge in sensitized animals reversed OVA-induced changes in NOS isoenzyme activities (n = 5, P < 0.001). In naive animals IL-10 was increased 1 h after LPS exposure (n = 5, P < 0.001), peaked at 3 h (n = 9, P < 0.001), and remained elevated above baseline at 18 h (n = 11, P < 0.05). In sensitized animals, IL-10 was not increased until 18 h after OVA challenge (n = 11, P < 0.001). Due to the rapid IL-10 increase in naive animals the released IL-10 is likely to be preformed; however, in sensitized animals the results are consistent with de novo production of IL-10. In the sensitized and OVA-challenged group, exposure to LPS 18 h after OVA produced a 3-fold increase in IL-10 at 3 h after LPS exposure (n = 5, P < 0.001). The time course and kinetics of IL-10 release in those animals was similar to that seen in naive rats. These results support our previous conclusions on the basis of in vivo studies using isoenzyme inhibitors and have shown LPS to be able to reverse OVA-induced changes in NOS isoenzyme activities during an established LAR. LPS-induced release of IL-10 is thought to play an important immunomodulatory role in this model.
Asunto(s)
Alérgenos/inmunología , Interleucina-10/metabolismo , Lipopolisacáridos/administración & dosificación , Óxido Nítrico Sintasa/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Administración por Inhalación , Alérgenos/administración & dosificación , Animales , Pruebas de Provocación Bronquial , Líquido del Lavado Bronquioalveolar/química , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Técnicas In Vitro , Lipopolisacáridos/inmunología , Masculino , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Ratas , Ratas Endogámicas , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/inmunología , Tráquea/efectos de los fármacos , Tráquea/inmunología , Tráquea/metabolismoRESUMEN
The in vivo role of nitric oxide in inflammatory cell migration, vascular permeability and the development of hyperresponsiveness to methacholine (MCh) was studied in rats 24 h following ovalbumin (OVA) challenge. The NO synthase (NOS) inhibitors N(G)-mono-methyl-L-arginine (L-NMMA; nonselective), aminoguanidine (two-fold inducible NOS-selective), N(omega)-nitro-L-arginine methyl ester (L-NAME; 2000-fold endothelial cell NOS-selective) or S-methyl-L-thiocitrulline (100-fold neuronal NOS-selective) were administered (100 mg x kg(-1) s.c.) to OVA-sensitized Piebald-Virol-Glaxo rats on 3 consecutive days during which they were challenged with allergen (1% OVA). Responses to inhaled MCh were measured in anaesthetized animals 24 h after OVA challenge. Cellular inflammation and vascular permeability were assessed using bronchoalveolar lavage (BAL) fluid collected 30 min after administration of Evans blue (50 mg x kg(-1) i.v.). OVA challenge in sensitized animals induced hyperresponsiveness to MCh, inflammatory cell influx and increased leakage of Evans blue into the BAL fluid (n=9, p<0.001). Aminoguanidine was effective in inhibiting the allergen-induced cellular influx and microvascular leakage (n=9, p<0.001) without altering responses to MCh. This effect was reserved by L-arginine. L-NAME (n=5, p<0.01) and S-methyl-L-thiocitrulline (n=6, p<0.001) further potentiated the allergen-induced hyperresponsiveness without altering cellular inflammation. L-NMMA attenuated both the OVA-induced cellular influx and Evans blue leakage (n=8, p<0.001) as well as further potentiating the hyperresponsiveness to MCh (p<0.05). From these studies, it is suggested that, in allergic Piebald-Virol-Glaxo rats, nitric oxide production by inducible nitric oxide synthase plays a role in the migration of inflammatory cells and increase in vascular permeability following allergen challenge, whereas nitric oxide produced by the constitutively expressed neuronal nitric oxide synthase limits hyperresponsiveness to methacholine.
Asunto(s)
Citrulina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Hipersensibilidad/inmunología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Tiourea/análogos & derivados , omega-N-Metilarginina/farmacología , Animales , Líquido del Lavado Bronquioalveolar , Broncoconstrictores , Citrulina/farmacología , Modelos Animales de Enfermedad , Masculino , Cloruro de Metacolina , Ratas , Tiourea/farmacologíaRESUMEN
The potential role of respiratory infections in altering the development of atopy and asthma is complex. Infections have been suggested to be effective in preventing the induction of T-helper 2-polarized allergen-specific immunity in early life, but also to exacerbate asthma in older, sensitized individuals. The mechanism(s) underlying these effects are poorly defined. The aim of this work was to determine the influence of lipopolysaccharide (LPS) exposure on the development of sensitization to allergen and the response to allergen challenge in vivo. Piebald-Virol-Glaxo rats were exposed to a single aerosol of LPS 1 d before or 1, 2, 4, 6, 8, or 10 d after sensitization with ovalbumin (OVA). On Day 11 animals were exposed to 1% OVA and responses to allergen were measured 24 h later, monitoring inflammatory cell influx and microvascular leakage into bronchoalveolar lavage (BAL) fluid as well as pulmonary responses to methacholine using the forced oscillation technique. Histologic analysis was included to complement the BAL results. Single aerosol exposure to LPS 1 d before and up to 4 d after intraperitoneal injection of OVA protected against the development of OVA-specific immunoglobulin (Ig) E. LPS exposure 6, 8, or 10 d after sensitization further exacerbated the OVA-induced cellular influx, resulting in neutrophilia and increased Evans Blue dye leakage with no effect on serum IgE levels. In addition, LPS abolished the OVA-induced hyperresponsiveness in sensitized animals when given 18 h after OVA challenge. This study demonstrates that exposure to LPS can modify the development of allergic inflammation in vivo by two independent mechanisms. Exposure early in the sensitization process, up to Day 6 after exposure to allergen, prevented allergen sensitization. Exposure to LPS after allergen challenge in sensitized animals abolished the hyperresponsiveness and modified the inflammatory cell influx characteristic of late-phase response to allergen.